While traditional studies of cellular ionizing radiation responses have focused on the direct deposition of energy in the nucleus, it is now recognized that cells also respond to extra-nuclear radiation damage, and even to extra-cellular radiation damage via the bystander effect. However, the differential signal transduction pathways regulating the responses to damage in different cellular compartments have not been well elucidated. This project seeks to harness the power of microarray profiling and functional genomics in conjunction with the single-cell / singleparticle microbeam irradiator in order to gain insight into the mechanisms of signaling between cellular compartments and between cells in response to radiation damage. Analysis of gene expression profiles induced by cytoplasmic and bystander irradiation of primary cells and a 3-D tissue model should lead to identification of molecular targets for modification of the radiation response. These will then be validated through the use of expression vectors, siRNA or chemical inhibitors. Modification of extracellular signaling may be especially attractive, both in cases of low dose exposures where not all cells suffer direct irradiation, and in limited field higher dose exposures, such as those given in radiotherapy, where late effects are known to occur outside the treatment field. Gene expression profiling will be used to advance our understanding of down-stream effector genes of the arachidonic acid cascade that may be important in mediating the bystander response. A series of four inter-related specific aims will address two main testable hypotheses. Ultimately, understanding the molecular basis for the communication of radiation damage within and among cells will advance our knowledge of the mechanisms of radiation response and the bystander effect.